RUI: Molecular evolution and regulation of the anthranilate branch point in plants

To defend themselves against herbivores, plants such as grape, maize, citrus, and strawberry emit a volatile called methyl anthranilate, which acts as a natural bird deterrent and attracts parasitic insects. While at least ten plant families produce methyl anthranilate, all plants synthesize the precursor, anthranilate, as an intermediate in the biosynthesis of the amino acid tryptophan (Trp), which is essential for plant growth and development.

The goal of this research is to understand how plants regulate anthranilate metabolism at the interface between amino acid synthesis and defense metabolism. Since methyl anthranilate is used commercially as a grape flavoring agent in the food and beverage industry and as an anti-avian spray for fields and crops, knowledge gained from these experiments may have implications in biotechnology and agriculture.

In addition to training diverse undergraduates in protein biochemistry, structural biology, systems biology, and plant genetics at a primarily undergraduate institution, this project will be used to develop a laboratory kit for elementary and middle school students in rural counties in Massachusetts and Vermont to investigate the importance of plant chemicals in defense against herbivores.

Plant chemical defenses are typically specialized metabolites that are synthesized from primary metabolites, like amino acids, or intermediates in primary metabolic pathways. Despite the fundamental role of amino acid biosynthesis in plant defense, the early steps of the Trp biosynthetic pathway remain to be biochemically characterized outside of microorganisms.

The Trp pathway starts by forming anthranilate, which is then conjugated to a phosphoribosyl sugar using a transferase (PAT1). Some plants siphon anthranilate away from the Trp pathway by methylating it, forming the volatile O-methyl anthranilate, while the Citrus family generates N-methyl anthranilate as a precursor to toxic acridone alkaloids. The goals of this research are to identify the molecular mechanisms governing anthranilate allocation to Trp versus methyl anthranilate biosynthesis and to trace the evolution of pathway regulation.

This project will take advantage of the wealth of available plant genome sequence data and use integrative approaches to investigate anthranilate-using enzymes. Aim 1 will identify the molecular determinants and evolution of PAT1 regulation using mutagenesis, activity assays, and X-ray crystallography. Aim 2 will trace the convergent evolution of anthranilate O- and N-methyltransferases using a structure-guided phylogenetic approach coupled with ex vivo metabolomics assays.

Aim 3 will further our understanding of anthranilate modulation using a systems-level approach to investigate phenotypic, metabolic, and gene expression changes in planta. This study will shine new light on the regulatory mechanisms of a fundamental amino acid biosynthetic pathway, as well as aid in our understanding of the evolution of convergent specialized metabolism.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.